固定化β-葡萄糖苷酶應用於黑豆漿中大豆異黃酮去醣基之研究

Abstract

大豆異黃酮是存在於大豆中的天然黃酮類化合物。研究發現，攝入足量的大豆異黃酮可以有效降低罹患骨質疏鬆症及婦女更年期綜合症的風險；在抗癌、防癌及預防心血管疾病等方面也有重要的功用。另有文獻指出，在人體內醣苷型的異黃酮不能直接被吸收，而是經由小腸中細菌產生的β- glucosidase水解去醣基後才進入血液，發揮各種生理作用。然而，人體小腸中的菌相基於個體差異活性會有很大差別，因此，若能直接攝入不帶醣基的苷元型異黃酮，則能有效提高其利用效率。本實驗之目的即是建立可將黑豆漿中大豆異黃酮醣苷轉化之固定化酵素系統。將β- glucosidase固定化於尼龍、纖維素以及凝膠等三種載體，實驗結果顯示每40 cm3載體上能固定超過40 mg之酵素，經酵素動力學測試後得到尼龍、纖維素以及凝膠系統其酵素動力學參數KM值分別為2.5042 ± 0.1901, 1.3826 ± 0.2043 和1.5889 ± 0.1152。以p-NPG作為基質測試固定化酵素活性後可知尼龍及纖維素系統具有較佳的反應穩定性。進一步使用HPLC分析固定化酵素應用於黑豆漿中異黃酮的轉化效率後得到纖維素系統為最佳反應體系，其最適作用溫度為50oC，並且可以在30分鐘之內利用40cm3含有酵素的載體將50 ml黑豆漿中的異黃酮全部轉化為去醣基化形式。在經過15次反應以及15天之後此系統仍然可維持其60%的酵素活性。

The studies of isoflavone have recently drawn tremendous attention due to its potential health-enhancing benefits, including reduction of cardiovascular disease, prevention of cancer, protection of osteoporosis, and antioxidant activities. Researches revealed that isoflavones in their aglycone forms exhibit higher biological activity and are more metabolically active that can be absorbed faster in greater amounts than their glycosides. This study is conducted to evaluate the feasibility of soymilk isoflavone conversion using an immobilized β-glucosidase enzyme system. β-glucosidase was immobilized on nylon pellets, cellulose beads, and sol-gel with the loading that over 40 mg of enzyme on 40cm3 of carriers. The KM value of these three enzymatic systems were determined to be 2.5042 ± 0.1901, 1.3826 ± 0.2043 and 1.5889 ± 0.1152, respectively. Nylon and cellulose system were proved more effective for the conversion of pNPG to p-nitrophenol. And with further determination, cellulose system exhibited a significant efficiency for isoflavone deglycosylation in black soymilk. The optimum temperature was 50 OC and complete reaction can be reached in 30 min. The amount changes of isoflavone in black soymilk after deglycosylation under different temperature were analyzed by HPLC. The reusability and storage stability were further verified. The enzymatic system can be used more than 15 cycles and 15 days which still retained 60% of the enzyme activity.